In 1993, Ajimura et al. isolated a temperature sensitive mutant of S. cerevisiae that was shown to be defective in initiation of meiotic recombination. This mutant, mre11-1, was sensitive to the radiomimetic agent methyl methanesulfonate (MMS) and showed a 10-fold increase in the level of mitotic recombination (Ajimura et al., Genetics 133:51–66,1993). Based on these properties, the MRE11 gene has been classified as belonging to same epistasis group as RAD50. A null mutant of MRE11 is unable to initiate meiosis, rendering the spores non-viable. The Mre11 protein has been shown to interact with the Rad50 protein to initiate double strand breaks in meiotic recombination (Johzuka K. and Ogawa H., Genetics 139:1521–1532, 1995). A new mutant allele, mre11S, was isolated and shown to block processing but not formation of double strand breaks (Nairz K. and Klein F., Genes and Dev. 11:2272–2290, 1997). Another mutant Mre11 allele which has been characterized, mre11-58, has been shown to contain two amino acid changes from the wild type protein. Interestingly, unlike mre11 null mutants, mre11-58 was proficient in formation of double strand breaks, but defective in processing of the DNA ends, indicating the involvement of Mre11 protein in exonucleolytic processing of double strand breaks during meiosis (Tsubouchi H. and Ogawa H., Mol. Cell. Biol. 18:260–268, 1998). This 3′ to 5′ exonuclease activity of Mre11 on double-stranded DNA either by itself, or in complex with Rad50 and Xrs2/p95 has been clearly established by two different groups (Paull T. and Gellert M., Mol. Cell 1:969–979,1998; Trujillo et al., J Biol Chem 273:21447–21450,1998). The exonuclease activity is observed only in the presence of Mn++. Mre11 also exhibits Mn++-dependent endonuclease activity on ssDNA (Trujillo et al., J Biol Chem 273:21447–21450, 1998) as well as on hairpin loops formed during V(D)J recombination (Paull T. and Gellert M., Mol. Cell 1:969–979, 1998).
The involvement of the MRE11/RAD50/XRS2 group of genes in non-homologous end joining (also known as non-homologous or illegitimate recombination) has also been well documented (Moore J K and Haber J E, Mol. Cell Biol. 16:2164–2173, 1996; Tsukamoto et al., Genetics 142:383–391, 1996; Wilson et al., Nucleic Acid Res 27:2655–2661, 1999; Lewis et al., Genetics 152:1513–1529, 1999). Furthermore, Mre11, along with Rad50 and Xrs2/p95, plays a critical role in the DNA damage response, as well as G2/M cell arrest following DNA damage, and DNA repair (Dolganov et al., Mol Cell Biol. 16: 4832–4841,1996; Carney et al., Cell 93:477–486,1998; Lee et al., Cell 94:399–409, 1998). Recently, Mre11 has been shown to be essential for the maintenance of chromosomal DNA (Yamaguchi-Iwai et al., Embo J. 18:6619–6629, 1999).
In summary, MRE11 is an important gene involved in meiotic and mitotic recombination, as well as homologous and non-homologous recombination. Thus, this single protein participates in multiple pathways that are often competing with each other such as double-strand break (DSB) formation in meiosis and DSB repair (via non-homologous end joining pathway) in mitosis. A very recent study by Furuse et al. employed two specific mutants of yeast Mre11 to elucidate this phenomenon (Furuse et al., EMBO J. 17:6412–6425; 1998). A point mutation in Mre11 (Asp16Ala) completely abolished the nuclease activity, without any change in DNA binding activity. This mutation also conferred MMS sensitivity to mitotic cells and caused them to accumulate unprocessed DSBs during meiosis. However, another mutant carrying a deletion of 49 C-terminal amino acids had almost wild-type levels of nuclease activity but reduced DNA binding activity. The mitotic phenotypes of this mutant were essentially unchanged, but the meiotic DSB formation was reduced dramatically. These results indicate the presence of two distinct functional domains on the Mre11 protein, an N-terminal region specifically involved in mitotic functions and a C-terminal 49 amino acid domain involved in the meiotic DSB formation. Thus, interactions of different domains with other proteins (such as Rad50 and Xrs2/P95) may be an underlying mechanism for the distinct roles of Mre11 in meiosis and mitosis (Usui et al., Cell 95:705–716, 1998). Whatever mechanisms may be involved, it is clear that either null or the N-terminal nuclease domain mutants of Mre11 are deficient in non-homologous end-joining.
Homologues of yeast MRE11 have been isolated from S. pombe (Tavassoli et al., Nucleic Acid Res. 23:383–388,1995), human (Petrini J H et al., Genomics 29:80–86, 1995; Chamankhah et al., Gene 225:107–116, 1998), and mouse (Xiao Y and Weaver D, Nucleic Acid Res. 25:2985–2991, 1997). Similarly, cDNA sequences encoding yeast Mre11-like proteins from Drosophila (Accession No. AF132144) Xenopus (Accession No. AF134569), Coprinus (Accession No. AF178433) and Arabidopsis (Accession No. AJ243822) have been deposited in the Genbank database.
Control of non-homologous end joining as well as mitotic and meiotic recombination by the modulation of Mre11, provides the means to modulate the efficiency with which heterologous nucleic acids are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.